PROJECT ABSTRACT Hepatitis A virus (HAV) is an unusual picornavirus (genus Hepatovirus) that is released without lysis from infected hepatocytes within small extracellular vesicles (EVs) resembling exosomes. These membrane-cloaked virions, or `quasi-enveloped' HAV (eHAV), lack virus-encoded glycoprotein peplomers on their surface, yet they are infectious and the only form of the virus found circulating in the blood of persons with acute hepatitis A. Numerous other `non-enveloped' viruses have been found to be released from cells as quasi-enveloped virions in EVs of varying size, making studies of eHAV relevant to a broad range of viral pathogens. This application proposes to investigate two aspects of lipid metabolism found to be essential for productive hepatovirus infection in a genome-wide forward genetic screen, and will test the following hypotheses: (i) that GD3 and possibly other gangliosides play a crucial role in cellular entry and the initiation of infection by both naked HAV and quasi- enveloped eHAV, by binding to and possibly triggering uncoating of the capsid within late endosomes or endolysosomes, and (ii) that HAV-infected cells and quasi-enveloped eHAV virions are highly enriched in sphingolipids with very long-chain fatty acid (VLCFA, C?22) tails, and that VLCFA synthesis is required for efficient production and release of infectious eHAV. In Aim 1, exceptional lipidomics expertise will be brought to bear on the question of which ganglioside species are necessary and sufficient for HAV and eHAV to enter cells and initiate infection, and will define the carbohydrate headgroups and acyl tail structures that are optimal for restoring the capacity of virus to infect CRISPR-derived ceramide glucosyltransferase (UGCG) knockout cells. The proposed experiments will also determine the impact of UGCG knockout on endocytosis and intracellular trafficking of HAV and eHAV, and the subcellular localization of exogenous gangliosides that restore the capacity of virus to enter these cells and initiate infection. Aim 2 will identify which ganglioside classes bind specifically to the naked HAV capsid, and whether ganglioside binding destabilizes its structure, lessening its thermostability at neutral or acidic pH, as a surrogate measure of uncoating. Aim 3 will characterize the role played by VLCFA in the hepatovirus lifecycle, and determine the degree to which VLCFA are enriched in infected cells, and in eHAV virions versus non-viral exosomes, and whether VLCFA are required for the production and cellular release of infectious eHAV, or for assembly of replication organelles involved in the synthesis of viral RNA. Additional experiments will determine whether and how HAV infection upregulates VLCFA synthetic flux, and whether this is essential for productive infection. By studying the role of lipids in hepatovirus replication, the proposed research will address an unexplored and neglected facet of the pathobiology of this important human pathogen.